Enterohepatic Helicobacter other than Helicobacter pylori
Beatriz Mateos-Muñoz
1, Julio Pérez-de-la-Serna
1, Antonio Ruiz-de-León
1, Blanca Serrano-Falcón
1, Sergio Casabona-Francés
1, Aurelio Velasco-Cerrudo
2and Enrique Rey-Díaz-Rubio
1Departments of
1Gastroenterology and
2Microbiology. Hospital Clínico San Carlos. Madrid, Spain
ABSTRACT
The Helicobacter genus includes Gram negative bacteria which were originally considered to belong to the Campylobacter genus.
They have been classified in a separate genus since 1989 because they have different biochemical characteristics, with more than 24 species having been identified and more still being studied.
H. pylori is the best known. It has an important etiopathogenic role in peptic ulcer disease and gastric cancer. Enterohepatic Helicobacters (EHH) other than H. pylori colonize the bowel, biliary tree and liver of animals and human beings with pathogenic potential. The difficulties existing to correctly isolate these microorganisms limit the description of their true prevalence and of the diseases they cause. Many studies have tried to discover the different clinical implications of EHH. Diseases like chronic liver disease, autoimmune hepatitis, hepatocarcinoma, autoimmune hepatobiliary disease, biliary lithiasis, cholangiocarcinoma and gallbladder cancer, Meckel´s diverticulum, acute appendicitis and inflammatory bowel disease have been related with different EHH species with different results, although their prevalence is greater than in healthy subjects. However, these data are currently not sufficient to draw definitive conclusions. Finally, the best known role of EHH in bowel disease is production of acute and chronic diarrhea pictures initially referred to as Campylobacter. H. pullorum has been identified in patients with acute gastroenteritis. The correct identification of EHH as producers of infectious gastroenteritis is found in its antibiotic susceptibility. It is generally macrolide- susceptible and quinolone-resistant.
Key words: Enterohepatic Helicobacter. Helicobacter pylori.
Helicobacter pullorum. Helicobacter hepaticus. Helicobacter bilis. Chronic liver disease. Inflammatory bowel disease. Acute gastroenteritis.
INTRODUCTION
The Helicobacter genus includes curved or helical flag- ellated Gram negative bacilli. It was first considered to belong to the Campylobacter genus. However, since 1989, due to its different biochemical characteristics, it has been classified in a separate genus (Table I). Since then, more than 24 species have been identified and 31 more are still being studied (1,2) (Fig. 1).
Within this genus, the best known species is H. pylori, a pathogen of the upper gastrointestinal tract. This species plays an important role in peptic ulcer disease and gastric cancer. Its microbiological characterization, definition of diagnostic techniques and approach to specific therapeutic strategies may have been the most relevant discovery in gastric disease in recent times (3). However, although the gastric Helicobacters have been the most studied, they only account for one third of the entire genus. The remain- ing two-thirds correspond to the so-called enterohepat- ic Helicobacter (EHH) (4) because they predominantly colonize the bowel, biliary tree and the liver of animals (Table II) and human beings. These microorganisms are less known because they are difficult to culture from sam- ples of these tissues and they require DNA hybridization and sequencing for their detection in most of the cases.
Development of new laboratory techniques such as fat- ty acid profile, mass spectrometry and polymerase chain reaction (PCR) have made it possible to identify these germs more specifically. They are also Gram negative bac- teria in spiral form that have a single polar flagellum. We could classify them into two groups, the first comprising flagellated bacteria which in turn have periplasmic cilia.
Received: 26-02-2012 Accepted:29-05-2013
Correspondence: Beatriz Mateos Muñoz. Department of Gastroenterology.
Hospital Clínico San Carlos. C/ Doctor Martín Lagos, s/n. 28040 Madrid e-mail: [email protected]
Mateos-Muñoz B, Pérez-de-la-Serna J, Ruiz-de-León A, Serrano- Falcón B, Casabona-Francés S, Velasco-Cerrudo A, Rey-Díaz- Rubio E. Enterohepatic Helicobacter other than Helicobacter pylori. Rev Esp Enferm Dig 2013;105:477-484.
POINT OF VIEW
The second subgroup lacks these and is structurally simi- lar to the microorganisms of the Campylobacter genus (4).
These similarities, together with the already-mentioned difficulties for their isolation, are an important limitation for the description of the pathogenic mechanism of these bacterias (5). The clinical interest of the EHHs comes from the growing number of publications of clinical cas- es as well as studies carried out on the involvement of these germs in liver, biliary and gastrointestinal diseases (Table III).
EHH AND HEPATOBILIARY DISEASES
Since the Helicobacter genus was identified, different bacteria capable of producing hepatobiliary diseases in animals have been characterized (6-24). H. hepaticus, H. rodentium, H. bilis and H. pullorum, among others, cause hepatitis and cholestasis pictures in mice and small mam- mals (6-15,21). In this way, research regarding the patho- genic influence of these microorganisms in humans has been an important point of interest during the last decade.
The serology, culture of tissue samples and especially the specific polymerase chain reaction for the 16s ribo- somal RNA of Helicobacter are the techniques by which H. hepaticus, H. pullorum and H. bilis have been identified in humans with hepatobiliary disease (25).
Chronic liver disease. Hepatic cirrhosis. Hepatitis C virus
Detection of DNA of Helicobacter spp. in the liver parenchyma has potentiated the approach to the hypothe- sis on a possible relationship between EHH infection and chronic liver disease in cirrhotic stage. Siringo et al. (26) carried out a case-control study that compared seropreva- lence of H. pylori in cirrhotic patients versus healthy blood donors. They obtained a positive serology in 76.5 % of the cases and 41.8 % of the controls (p < 0.0005). How- ever, the multivariate analysis revealed that these data were affected by other variables such as age and sex.
Fig. 1. Microphotograph of Helicobacter pullorum in a sample of stools of a male patient with acute gastroenteritis. Gram stain (1000x).
Table I. Enterohepatic Helicobacter. Microbiological characteristics. Similarities and differences with the Campylobacter genus (modified Schauer) (4)
Catalase Urease Growth
at 42 ºC
Number of flagella
Covered flagella Periplasmic cilia
Campylobacter genus + - + Unipolar + -
H. hepaticus + + - Bipolar + -
H. cinaedi + - - Unipolar + -
H. fennelliae + - - Bipolar + -
H. canis - - + Bipolar + -
H. parmetensis + - + Bipolar + -
H. pullorum + - + Unipolar - -
H. candensis + - + Uni/bip. - -
H. rodentium + - + Bipolar - -
H. typhlonicus + - - Bipolar + -
H. cholecystus + - + Unipolar + -
H. mesocricetorum + - + Bipolar - -
H. muridarum + + - Bipolar + +
H. rappini - + + Bipolar + +
H. bilis + + + Bipolar + +
H. trogontum + + + Bipolar + +
Table II. Enterohepatic Helicobacter: Isolations in animals and related pictures in them
Species Host Isolation site Disease
H. pullorum (6-8) Chicken and hen Gastrointestinal tract and liver Gastroenteritis Mild acute hepatitis H. hepaticus (9-12) Mouse Liver, bile duct and gall bladder Chronic hepatitis
Typhlitis. IBD
Hepatocellular tumors H. bilis (13-15) Mouse and cat Gastrointestinal tract and liver Chronic hepatitis
Typhlitis Gastroenteritis H. rappini (16,17) Sheep, dogs and mice Liver, stomach
Ovine abortions
Gastroenteritis
Abortions by hepatic necrosis H. cinaedi (18) Hamster, Rhesus monkeys and dogs Gastrointestinal tract Gastroenteritis
Bacteremia H. fenneliae (19) Dogs and macaques Gastrointestinal tract Gastroenteritis
H. canis (20) Dogs, cats Stools Gastroenteritis
H. rodentium (21) Mouse Liver, gastrointestinal tract Acute cholangitis
Typhlitis
H. muridarum (22) Mouse Gastrointestinal tract and
stomach
Gastroenteritis Gastritis
H. trogotum (23) Rats Bile ducts Hepatitis
H. aurati (24) Hamster Gastrointestinal tract and
stomach
Chronic gastritis Intestinal metaplasia Typhlitis
Table III. Helicobacter related with different diseases in humans
Species Hepatobiliary disease Pancreatic disease Bowel disease
H. pylori Chronic liver disease (26,27) HCC (30-40)
Pancreatic tumors (61-66) Meckel´s diverticulum (77,78) Acute appendicitis (79) H. hepaticus Chronic liver disease (27)
HCC (30-40) PSC and PBC (45) Cholelitiasis (48,50,52) H. pullorum Liver disease by HCV (29)
PSC and PBC (45) Cholelitiasis (49)
Meckel´s diverticulum (76) Acute appendicitis (76) IBD (81-84)
Acute gastroenteritis (74,86)
Post-infectious irritable bowel syndrome (74) H. bilis Liver disease by HCV (29)
HCC (30-39) PSC and PBC (45) Cholelitiasis (47,48,51) Cholangiocarcinoma (47,48,51) Acute and chronic cholecistitis (51) H. ganmani Cholelitiasis (51)
Cholangiocarcinoma (51)
Acute and chronic cholecystitis (51)
H. cinaedi Proctitis and proctocolitis (75)
Enteritis (4)
H. rappini Chronic diarrhea (75)
H. fennelliae Proctitis and proctocolitis (75)
Enteritis (4)
Because of the possibility that the serology of H. pylori could have a cross-reactivity with IgG of other bacteria of the genus, Nilsson et al. (27) studied the seroprevalence of H. pylori and H. hepaticus in 144 patients with chronic liver disease of different etiologies. They found a similar seroprevalence of both species (44 vs. 39 %), which is also similar to that existing in the general population (48 vs.
46 %). At the same time, they discovered a specific immune reaction against H. hepaticus, with greater concentration of antibodies in 26 % of the cases. However, this higher titer did not seem to be related to baseline hepatic disease.
A possible relationship between EHH and hepatitis C virus (HCV) has also been proposed. In this sense, Ponzetto et al. (28) determined that the seroprevalence of Helicobacter spp. in cirrhotic patients with positive HCV is 77 %, while it is about 59 % in healthy controls (p <
0.004). Lönngren et al. (29) described seroprevalences of 18 % for H. pullorum and 8 % for H. bilis in positive HCV patients versus 0 % in healthy subjects. The possibility that EHH may contribute to progression of chronic liver dis- ease due to this virus has been analyzed in a stratified way, classifying the study subjects into four groups: Healthy patients, patients with chronic hepatitis, cirrhotic patients and patients with hepatocellular carcinoma (HCC). Pres- ence of Helicobacter spp. DNA in each one of the groups was studied. A total of 68 % of the patients with hepat- ic cirrhosis and 90 % of those with HCC had DNA of this bacteria genus compared to 4.2 % of patients with chronic hepatitis and 3.5 % of the controls, which means a statistically significant difference regarding the pooled analysis of the data from the first two versus the latter two groups (p < 0.0001) (30). However, unfortunately the results are not very conclusive since these studies have been performed with a small number of patients and have potential confounding factors such as the course, per se, of chronic viral liver disease. Thus, their conclusions must be considered with caution until new studies are published.
Hepatocarcinoma
Based on the known gastric carcinogenesis produced by H. pylori and motivated by the good therapeutic response to it, the possibility has been proposed that EHH would have a role in the appearance of HCC. There are 10 case-control studies on this association (30-39). All of them, except one (39), describe a significantly greater presence of EHH DNA in the liver tissue of patients with HCC than in healthy subjects (40). Although these results are remarkable, the heterogeneity of the samples and the study design as well as the presence of hepatotropic virus infection prevent the acquisition of definitive conclusions.
Different hypotheses have been postulated on the mecha- nisms of carcinogenesis that the EHH could have, whether indirectly by the production of chronic inflammatory cyto- kine that contributes to cellular damage (41), or directly
by DNAsa activity of cytolytic toxins that are produced by these germs (42). On the other hand, it should be stated that the fact that DNA of these microorganisms can be detected but not isolated in a culture casts some doubts on the existence of a real infection. However, the results indicate that they are a more than sufficient motivation to continue this line of research.
Autoimmune hepatobiliary disease
In hepatic biopsies of patients with primary sclerosing cholangitis (PSC) and primary biliary cirrhosis (PBC), both the presence of EHH DNA (43) and bacteria of this genus in Kupffer cells have been detected (44). Based on these results, Nilsson et al. (45) carried out a study of the seroprevalence of H. pullorum, H. bilis and H. hepaticus in patients with PSC, finding that each one of them were at 38 %, 22 %, 25 %, respectively and that this prevalence was significantly greater than in the healthy controls (p <
0.05; p < 0.001 and p < 0.001, respectively) in every case.
Equally, considering patients with autoimmune hepatitis as cases, a seroprevalence of 30 % was obtained for H. pullo- rum, 22 % for H. bilis and 12 % for H. hepaticus, all being statistically significant (p < 0.001 in all of them) compared to the controls. In the cases with positive EHH, there was a statistically significant tendency towards higher levels of alkaline phosphatase and lengthening of prothrombin activity time than in healthy subjects (p < 0.0001 and p <
0.0003), but not in the total bilirubin (43). Sample size of these studies is small so that the results are not conclusive.
However, considering that autoimmune diseases are caused by genetic as well as environmental factors, it is proposed that EHH could play an important role in these mechanisms on the hepatic level that still must somehow be elucidated.
Biliary disease
Helicobacter spp. has been isolated from the bile of
subjects with different diseases as well as from of healthy
subjects (46). In these cases, the possibility of ascending
colonization from the duodenum to the bile ducts and gall-
bladder by these microorganisms is considered. Its surviv-
al in such an inhospitable environment as the bile could
be favored by the peptidase enzymes that these germs
have (25). The role that these bacteria could play in the
lithogenesis has been investigated in different projects,
in which the presence of H. bilis (47,48), H. pullorum
(49) H. hepaticus (48,50) and H. ganmani (51) has been
studied with different techniques (PCR, serology and cul-
ture, among others) and in different samples (blood, bile,
gallbladder) in patients with cholelithiasis and in control
subjects. The results, although very heterogeneous, show
greater prevalence of lithiasis in EHH infected patients
in every case. Specifically, H. hepaticus infection triples
the possibility of having cholelithiasis (OR 3.13, 95 % CI 1.20-8.19) (52).
At the same time, it was observed that greater bile acid- ity meant greater survival of these species, so that condi- tions such as cholecystitis and obstructive biliary disease in which the biliary pH decreases would favor secondary bacterial overgrowth and would collaborate in the infec- tion of the biliary system (25). In this regards, several studies have been made in an attempt to know the role of EHH in acute and chronic cholecystitis (15,51,53-56). The results obtained are very heterogeneous, with a prevalence of EHH in cholecystectomy specimens by these conditions of 39 % in Chile (15), while in Germany it is 2 % (51).
These discrepancies could be based on the geographic differences with different prevalences of EHH, although the differences in the diagnostic methods used and in the heterogeneity of the study design should also be stressed.
There is also a hypothesis that relates EHH with cholan- giocarcinoma and gallbladder cancer. In this way, already previously-mentioned studies (47,48,51,53,54) and others (57-60) evaluated the presence of Helicobacter DNA by PCR in cholecystectomy samples. The results, as in the previous point, were very diverse so that it is not possible to draw definitive conclusions.
EHH AND PANCREATIC DISEASE
In recent years, there have been different hypotheses on the participation of bacterias of the Helicobacter genus in pancreatic disease. The most important ones mention its possible oncogenic role in pancreatic cancer. Sever- al studies are found on seroprevalence of H. pylori in patients with pancreatic tumors (61-66) that show a sig- nificant relationship with an OR of 1,379 (95 % CI 1,083- 1,796) regarding the controls. However, there are only two projects that study this relationship using molecular techniques (67,68): Jenawski et al. (67) that analyzes the presence of Helicobacter spp. DNA in pancreatic juice and tissue of patients with pancreatic adenocarcinoma without obtaining a positive result. In addition, Nilsson et al. (68) also studied this relationship by PCR, demonstrating the existence of DNA of these microorganisms in tissue sam- ples: In 75 % of the cases of exocrine tumors, in 57 % of the neuroendocrines and in 60 % of the samples obtained from patients with multiple neoplasia syndrome. Differ- ent oncogenic mechanisms by which Helicobacter could produce pancreatic cancer have been postulated, although none have been molecularly demonstrated (69). It should also be pointed out that as in the case of hepatobilary dis- ease, the Helicobacter genus has been related with differ- ent autoimmune conditions. Autoimmune pancreatitis is a condition that has been linked to these bacteria but there are no studies with results that support it (68). However, there are different pathogenic hypotheses that principally collect cross-reactivity mechanisms (70).
EHH AND INTESTINAL DISEASE
Multiple species of the Helicobacter genus have been isolated in the gastrointestinal tract in both asymptomatic animals and in those with gastritis or enteritis. Thus, H.
pullorum, H cinaedi, H. canis, H. bilis and H. muridarum have been described as responsible for this type of condition in poultry and domestic animals (4). In humans, these micro- organisms have been detected by PCR in stools of patients with acute gastroenteritis (8,71-75) and have been related with cases of Meckel´s diverticulum (76-78), acute appen- dicitis (79) and with inflammatory bowel disease (80-85).
Meckel´s diverticulum
Based on the fact that it is not uncommon to find ecto- pic gastric mucosa in Meckel´s diverticulum, Bemelman et al. (77) and Tuzum et al. (78) studied the possibility that this mucosa would be infected by H. pylori and that this would collaborate in the clinical manifestations of the diverticulum. These authors investigated the presence of the microorganism by microscope and PCR of the surgi- cal specimens, respectively, but the results were not very encouraging. Bemelman et al. (77) obtained 1 case out of 18 diverticula with positive ectopic gastric mucosa for H. pylori and Tuzun et al. (78) none out of 12 cases. In 2011, Karagin et al. published a study on the presence of EHH in patients with Meckel´s diverticulum (76). They found DNA of H. pullorum by PCR in 3 % of the cases.
However, they were not able to demonstrate the presence of the germ by immunohistochemistry study.
Acute appendicitis
The role of bacterias of the Helicobacter genus in acute appendicitis has also been discussed. There are few stud- ies published about this. Paredes et al. (79) conducted a case-control study analyzing the presence of H. pylori antigen in stools and in the culture of the appendecto- my samples. They observed antigens in stools in 35 % of patients, finding that 71.4 % of the cultures were also positive. However, this was not statistically significant in comparison with the controls. Karagin et al. (76) analyzed the presence of EHH in similar samples by PCR but none of the cases were positive.
Inflammatory bowel disease
Ulcerative colitis and Crohn´s disease are two condi-
tions for which an effort has been made to relate them with
EHH. Due to the participation of the genetic and environ-
mental factors, among them microbiological ones, in the
pathogenesis of inflammatory bowel disease (IBD), the
possible influence of these microorganisms in its devel- opment was postulated. It had been demonstrated in the laboratory that bacteria of the Helicobacter genus would produce IBD in immunodepressed mice (85), so that its presence in human patients suffering IBD was also stud- ied. The results of the studies on this relation are hetero- geneous. Thus, in the earliest studies on the subject, it was not possible to detect DNA of any EHH in colonic mucosa samples (80). However, more recent studies have found a statistically significant relation between EHH and IBD (83,84). Laharie et al. (81) analyzed the presence of DNA of these bacteria by PCR in a total of 44 sam- ples obtained by colonoscopy in patients with Crohn´s disease and healthy controls. They defined a prevalence of 12 % in the cases compared to 4 % in the controls. In patients with ulcerative colitis, in a study with a similar design, Thomson et al. (82) found a prevalence of 61 % in patients compared to 10 % in healthy controls. However, these results must be treated with caution because of the same sample size of the studies published and especially the existing difficulty to obtain homogeneous samples of patients.
Acute infectious gastroenteritis
Finally, perhaps the best known role of EHH in bowel disease is the production of acute and chronic diarrhea.
Until a few years ago, enteritis caused by these microor- ganisms were included within those caused by the Cam- pylobacter genus due to their microscopic similarity (86).
However, the development of new laboratory techniques as the fatty acid profile (87), mass spectrometry and the PCR (8) have made it possible to identify the germs more specifically. As these techniques become more general- ized, more will become known on the real prevalence of these bacteria in the population (8). EHHs have been isolated in the digestive tract of poultry and domestic ani- mals (88,89) and H. pullorum was designated as a sepa- rate species, on the basis of sequencing of the 16S rRNA gene (7). H. pullorum has been isolated in cecal samples from broiler chickens with subclinical infection, in the liver and intestinal content of laying hens with vibrionic hepatitis and in human beings with gastroenteritis (7,8).
Furthermore, elevation of hepatic enzymes and hepato- megaly was observed in one patient with diarrhea (73). It has been demonstrated in vitro that H. pullorum survives more than 36h in water (90). Therefore, although the epi- demiological chain has not been clearly defined (72), there appears to be a basis to consider food borne transmission of the gastroenteritis of H. pullorum as occurs with the Campylobacter species (4). Generally, they cause watery diarrhea with rectal bleeding in which it is believed that a cytotoxic toxin that has been isolated in certain cases of H. pullorum diarrhea could be involved, although not in other EHHs (86). In our center, where Helicobacter pullo-
rum is being investigated systematically since April 2010, 9 cases that affected patients with a wide age spectrum have been identified (74). The clinical picture of watery diarrhea was accompanied by rectal bleeding in 33 % of the cases, abdominal pain in 55 % and weight loss in 44 %. In the evolution, 55 % of the patients reported alternating bowel habit and existence of periods of abdominal pain. Antimi- crobial susceptibility of the strains was quite uniform, all of them susceptible to macrolides (erythromycin and azith- romycin) and to amoxicillin-clavulanate, while 80 % were quinolone-resistant (ciprofloxacin and levofloxacin).
Two cases of chronic diarrhea apparently caused by H. rappini (75) have been described. One of them was associated to fever and headache, with background of contact with infected domestic animals and another with- out fever or known contact with animals. Both responded effectively to erythromycin. Other species of Helicobacter such as H. cinaedi (from Latin for “of a homosexual”) or H. fennelliae were isolated from rectal samples of male homosexuals and also in asymptomatic individuals and individuals with proctitis, proctocolitis, and enteritis.
Although H. cinaedi has been described as a cause of acute diarrhea in otherwise healthy subjects, it more often causes bacteremia in patients with immunodeficiency (4).
It could be noted that the greatest relevance of the cor- rect identification of EHH as producers of infectious gas- troenteritis is found in the antibiotic susceptibility of these microorganisms, since they generally present resistance to quinolones (85 % for ciprofloxacin, 75 % for levofloxacin) and they have a bimodal behavior regarding tetracyclines (resistant in 76 % of the cases) and macrolides (suscepti- ble in 85 %) (91). Furthermore, as has been stated, EHH should be investigated in cases of chronic diarrhea and, in our opinion, in patients with diarrhea and symptoms con- sistent with irritable bowel syndrome of possible post-in- fectious etiology (74).
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